In many areas of research, the ability to separate animals or other large biological particles according to their phenotype is desirable. For example, there are now thousands of mutant Drosophila strains available. In fact, a project is underway to isolate a mutation in every Drosophila gene. However, in a breeding population three-quarters of the animals carry at least one normal chromosome and only one-quarter carry two mutant chromosomes. The ability to separate populations of mutant embryos from their normal siblings would greatly enhance the molecular and biochemical studies of these newly identified and uncharacterized genes.
Present technology in cell sorting is limited to the isolation of individual cells. Through the use of a flow cytometer, cells are sorted on the basis of their levels of fluorescence. The cells are placed in a laminar stream of liquid and flowed through a small opening where a jet in air is formed. When this jet is mechanically vibrated, it breaks into regularly spaced drops, such that there is approximately one cell per drop. A cell is then sorted or isolated by putting an electric charge on the droplet of water, which can then be deflected according to the charge. However, large biological particles, such as embryos or small animals, are heavy and difficult to deflect accurately by such a method. The present invention addresses this problem by eliminating the need for deflection.
Flow cytometers for use in sorting single cells are described in a number of publications. Exemplary is U.S. Pat. No. 5,880,474, issued Mar. 9, 1999, and the references cited therein. Krasnow et al. (1991) Science 251:81-85 describes the use of a conventional cell sorter to analyze the cells of a dissociated Drosophila embryo.
Other particle sorters have been described in the art. For example, Satake et al., U.S. Pat. No. 5,713,473, issued Feb. 3, 1998 describes a conveyer belt type sorter for beans.
Other art in this field is evident in the COPAS(trademark) fluorescence based sorter (manufactured and sold by Union Biometrica, Somerville, Mass.). Unlike the present invention, the COPAS sorter uses a fluid switch to interrupt and redirect particle flow. See International Patent application WO 00/11449.
An automated particle sorter is provided, which allows the separation of large multicellular biological particles, including embryos, small organisms and the like. The particle sorter provides a means of sorting multicellular aggregates that are too large to be sorted with an electrostatic deflection flow cytometer. The particle sorter comprises a fluid flow path, which places single biological particles in an optical cuvette. An exciting light irradiation system directs a source of light through the cuvette. The light excites a fluorescent substance present on the particle, and the emitted light is detected by a light detection apparatus comprising at least two detection elements for measuring the fluorescence emitted from the fluorescent substance. A light separation element, such as a dichroic mirror, is employed to separate the fluorescent light from the exciting light. A data processor compares the signal received from the fluorescent light; and from the background autofluorescent signal, and according to pre-set parameters, controls a mechanical switch that alters the position of a collection conduit between two set points. The conduit is composed of at least two tubes separated by a very thin central wall, e.g. a membrane. Sorting is achieved by moving the appropriate tube under the fluid stream. The tubes can, in turn lead to other collection vessels.